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Fig. 4 | Journal of Neuroinflammation

Fig. 4

From: Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain

Fig. 4

C1q reactivity is present in neonatal microglia of C1qa FL/FL :Cx3cr1 CreERT2 pups, but is decreased at 1 month and continues to decline with age in C1qa FL/FL :Cx3cr1 CreERT2. Representative images of C1q immunostaining (red) in the hippocampus (a–h) and cortex (j–m) of C1q FL/FL (a, b), WT (c, d), or C1q FL/FL :Cx3cr1 CreERT2 (e–h, j–m) at different ages as noted in the panels. Identical exposure time and illumination settings were used for each image in (a–h) and similarly were identical for the images in (j–m). Merged images of C1q immunostaining (red) and YFP (green) showing C1q colocalization with microglia (yellow, arrows) or progressive lack of C1q (YFP green only, arrowheads) in C1q FL/FL Cx3cr1 CreERT2 mice with age (j–m). Average of the mean intensity of C1q immunostaining in the molecular layer (*) of C1qa FL/FL (grasy) or C1qa FL/FL:Cx3cr1 CreERT2 (black) at different ages (1 month, n = 4; 1.5 months, n = 1; 2 months, n = 2; 5 months, n = 3; 7 months, n = 2), respectively (i). p values obtained from data from 3–4 mice at 1 and 5 months by ANOVA single factor analysis are p = 0.02 and 0.005, respectively. Quantification of the percentage of total microglia that were C1q positive in the cortex of C1qa FL/FL:Cx3cr1 CreERT2 mice (n). Percentage of C1q-positive microglia per animal was obtained by averaging five images per section per mouse. Bar represent average % of C1q-positive microglia in n mice per age (1 month, n = 4; 1.5 months, n = 1; 2 months, n = 2; 5 months, n = 3; 7 months, n = 2). Brain extracts (60 ug per lane) from C1qa FL/FL and C1qa FL/FL:Cx3cr1 CreERT2 littermates at 1 to 7 months of age, C1qKO at 5 m, and wild type (WT) plasma (1 ul) (o), were run as in Fig. 1. Representative of 3–9 animals per genotype. C1qa, C1qb, and C1qc mRNA relative to Hprt (p) in the brain from C1qa FL/FL (FL), C1qa FL/FL:Cx3cr1 CreERT2 at 3, 7, or 14 days after treatment with vehicle (V) or tamoxifen (T), C1qaKO (KO), and wild type (WT) mice by qPCR. Cells isolated from the brains of 2-day-old C1qa FL/FL :Cx3cr1 CreERT2 (YFP+) and C1qa FL/FL (YFP−) mice were stained with anti-CD11b, sorted by CD11b and YFP and subsequently stained with anti-C1q (red) and DAPI (blue) (q–s). Representative pictures of microglia (CD11b+) that are (q) Cx3cr1 CreERT2 (YFP+) show similar C1q staining compared to (r) C1qa FL/FL (YFP−) microglia (CD11b+). No C1q is present in populations negative for CD11b and YFP (neurons, astrocytes) (s). Scale bar 100 μm (a–h) or 50 μm (j–m,q–s)

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